Visualization and isolation of live primordial germ cells aimed at cell-mediated gene transfer in rainbow trout

Abstract

To develop cell mediated gene transfer in fish, establishment of a cell line that has an ability to differentiate into germ cells is needed. Although the several fish ES-like cell lines have been established, none of them can contribute to the germline in vivo. Therefore, we have chosen primordial germ cells (PGCs), the progenitor cells of the germline, as an initial material for in vitro cell culture. In this chapter, our current research on the techniques to identify, visualize, and isolate live PGCs of rainbow trout are introduced. Firstly, rainbow trout vasa-like gene (RtVLG) was characterized and the applicability of its expression as an indicator of PGCs was confirmed. Secondly, transgenic rainbow trout lines carrying GFP gene driven by the RtVLG regulatory regions were produced. In this transgenic embryo, PGCs were visualized by strong green fluorescence. Finally, a technique to isolate pure population of GFP-labeled PGCs from transgenic embryos by flow cytometer was developed. We are presently attempting to establish cell lines derived from trout PGCs isolated in our laboratory as an alternative for fish ES cells.